1.1 Blotting

See Table 20.1.

1.2 Polymerase Chain Reaction (PCR)

• 
  

  aim: amplify specific DNA/RNA sequences in a sample with many DNA/RNA fragments

  
  
• 
  

  if RNA is being amplified, it must first be transcribed into complementary DNA

  
  
• 
  

  steps:

  
  
  
  
  1. 
     

     1. Attach primer onto target DNA

     
     
  2. 
     

     2. Taq polymerase adds nucleotides onto new DNA strand

     
     
  3. 
     

     3. Process repeated to produce many DNA copies

  
  

1.3 DNA Microarrays

• 
  

  aim: allows simultaneous analysis of thousands of gene expressions

  
  
• 
  

  microarray is a commercially produced collection of fluorescently labelled DNA short oligonucleotides

  
  
• 
  

  steps:

  
  
  
  
  1. 
     

     1. Isolate RNA from cells

     
     
  2. 
     

     2. Translate RNA into complementary DNA

     
     
  3. 
     

     3. Hybridise cDNA onto microarray oligonucleotides

  
  

1.4 Bioinformatics

• 
  

  definition: computing method to store, distribute and analyse large reported DNA information

  
  
• 
  

  researchers report DNA fragment information into databanks

  
  
• 
  

  databanks are accessible via the Internet

1.5 Proteomics

• 
  

  definition: the study of the structure and function of proteins

2.1 Cancer

• 
  

  most cancers are caused by mutations in somatic cells (rather than germ cells)

  
  
• 
  

  it takes many years to accumulate the mutations (mutations need at least 20 years to cause cancer)

2.2 Cell Cycle Control Checkpoints

• 
  

  DNA damage checkpoints:

  
  
  
  
  1. 
     

     ◦ where: S phase, G1, G2

     
     
  2. 
     

     ◦ damage detected → cyclin-dependent kinase 2 (CDK2) inhibited → cell cycle progression stopped

     
     
  3. 
     

     ◦ damage not repairable → apoptosis

  
  
  
  
• 
  

  spindle checkpoint:

  
  
  
  
  1. 
     

     ◦ where: metaphase

     
     
  2. 
     

     ◦ spindle fibres fail to attach to kinetochores → apoptosis

  
  

2.3 Signalling Proteins

• 
  

  signalling proteins include growth factors (+ receptors), signal transduction proteins and transcription factors

  
  
• 
  

  gain-of-function in these proteins can lead to cancer

  
  
• 
  

  mutated alleles usually dominant

2.4 Cell Cycle Control Proteins

• 
  

  cell cycle control proteins are usually tumour-suppressor proteins

  
  
• 
  

  loss of function in these proteins can lead to cancer

  
  
• 
  

  mutated alleles are usually recessive

  
  
• 
  

  adenomatous polyposis coli (APC):

  
  
  
  
  1. 
     

     ◦ function:

     
     
     
     
     1. 
        

        ▪ activates transcription factor Myc → transcribes genes pushing movement from G1 to S phase

     
     
     
     
  2. 
     

     ◦ mutation:

     
     
     
     
     1. 
        

        ▪ if APC mutated → inappropriately activates Myc → uncontrolled cell proliferation

        
        
     2. 
        

        ▪ requires both APC alleles to be mutated for the protein to fail

     
     

  
  
  
  
• 
  

  p53:

  
  
  
  
  1. 
     

     ◦ functions:

     
     
     
     
     1. 
        

        ▪ detects DNA damage

        
        
     2. 
        

        ▪ blocks CDK2

        
        
     3. 
        

        ▪ activates apoptosis

     
     
     
     
  2. 
     

     ◦ mutation:

     
     
     
     
     1. 
        

        ▪ requires both p53 alleles to be mutated for the protein to fail

        
        
     2. 
        

        ▪ fifty percent of cancers have mutations in p53

     
     

  
  
  
  
• 
  

  ataxia telangiectasia mutated:

  
  
  
  
  1. 
     

     ◦ detects DNA damage

     
     
  2. 
     

     ◦ stops cell cycle

     
     
  3. 
     

     ◦ maintains normal telomere length: prevents chromosome shortening with DNA replication

  
  

2.5 Oncogenic Viruses

• 
  

  some viruses contain proto-oncogenes and oncogenes

  
  
• 
  

  DNA viruses:

  
  
  
  
  1. 
     

     ◦ need oncogenes for their own viral survival

     
     
  2. 
     

     ◦ example: human papillomavirus family

  
  
  
  
• 
  

  RNA viruses:

  
  
  
  
  1. 
     

     ◦ retrovirus enters host → makes DNA copies from viral RNA → DNA copies inserted into host’s DNA for viral replication → oncogenes produced

     
     
  2. 
     

     □ examples:

     
     
     
     
     1. 
        

        — Harvey sarcoma virus:

     
     
     
     
     1. 
        

        – contains Ha-ras gene (differs from the human ras gene by a single-point mutation)

        
        
     2. 
        

        – Ha-ras overexpression → bladder cancer

     
     
     
     
  3. 
     

     — Rous sarcoma virus

     
     
     
     
     1. 
        

        – produces v-Src protein, which is a constitutively active mutant of human c-Src protein

        
        
     2. 
        

        – leads to continuous phosphorylation of proteins